Bacterial expression of apolipoprotein (apo) B cDNA constructs has been used to map a series of monoclonal antibodies (mAbs)
to apoB by immunoblotting. In some cases assignments have been confirmed ...and refined by (i) semipurification of expressed
protein, CNBr digestion, and assignment of the immunoreactive fragments; (ii) controlled digestion of the cDNA with the exonuclease
Bal31 and bacterial expression of the truncated proteins that result; or (iii) expression of specific segments of cDNA amplified
by the polymerase chain reaction. Forty mAbs were mapped to a minimum of 17 separate determinants on apoB. Tryptic fragments
have been used to confirm the epitope assignments. In addition, this approach in conjunction with immunoassay, enables some
deductions to be made about the trypsin-accessible regions in low density lipoprotein (LDL). The cleavage pattern obtained
predicts retention of structure in the cysteine-rich domain of the amino terminus and also in the LDL receptor binding region.
Trypsinized LDL was shown to bind to the LDL receptor by an authentic process, using monoclonal antibodies as competing ligands.
In conjunction with the previous paper (Milne, R. W., Theolis, R., Maurice, R., Pease, R. J., Weech, P. K., Rassart, E., Fruchart,
J.-C., Scott, J., and Marcel, Y. L. (1989) J. Biol. Chem. 265, 19754-19760) the mapped mAbs have been used to define the receptor-binding
domain of apoB100 in LDL.
Monoclonal antibody 2E8 is specific for an epitope that coincides with the binding site of the low density lipoprotein receptor (LDLR) on human apoE. Its reactivity with apoE variants resembles that ...of the LDLR: it binds well with apoE3 and poorly with apoE2. The heavy chain complementarity-determining region (CDRH) 2 of 2E8 shows homology to the ligand-binding domain of the LDLR. To define better the structural basis of the 2E8/apoE interaction and particularly the role of electrostatic interactions, we generated and characterized a panel of 2E8 variants. Replacement of acidic residues in the 2E8 CDRHs showed that Asp(52), Glu(53), and Asp(56) are essential for high-affinity binding. Although Asp(31) (CDRH1), Glu(58) (CDRH2), and Asp(97) (CDRH3) did not appear to be critical, the Asp(97) --> Ala variant acquired reactivity with apoE2. A Thr(57) --> Glu substitution increased affinity for both apoE3 and apoE2. The affinities of wild-type 2E8 and variants for apoE varied inversely with ionic strength, suggesting that electrostatic forces contribute to both antigen binding and isoform specificity. We propose a model of the 2E8.apoE immune complex that is based on the 2E8 and apoE crystal structures and that is consistent with the apoE-binding properties of wild-type 2E8 and its variants. Given the similarity between the LDLR and 2E8 in terms of specificity, the LDLR/ligand interaction may also have an important electrostatic component.
Monoclonal antibody 2E8 is specific for an epitope that coincides with the binding site of the low density lipoprotein receptor (LDLR) on human apoE. Its reactivity with apoE variants resembles that ...of the LDLR: it binds well with apoE3 and poorly with apoE2. The heavy chain complementarity-determining region (CDRH) 2 of 2E8 shows homology to the ligand-binding domain of the LDLR. To define better the structural basis of the 2E8/apoE interaction and particularly the role of electrostatic interactions, we generated and characterized a panel of 2E8 variants. Replacement of acidic residues in the 2E8 CDRHs showed that Asp52, Glu53, and Asp56 are essential for high-affinity binding. Although Asp31 (CDRH1), Glu58 (CDRH2), and Asp97 (CDRH3) did not appear to be critical, the Asp97 → Ala variant acquired reactivity with apoE2. A Thr57 → Glu substitution increased affinity for both apoE3 and apoE2. The affinities of wild-type 2E8 and variants for apoE varied inversely with ionic strength, suggesting that electrostatic forces contribute to both antigen binding and isoform specificity. We propose a model of the 2E8·apoE immune complex that is based on the 2E8 and apoE crystal structures and that is consistent with the apoE-binding properties of wild-type 2E8 and its variants. Given the similarity between the LDLR and 2E8 in terms of specificity, the LDLR/ligand interaction may also have an important electrostatic component.
TwoATM variants and breast cancer risk Thompson, Deborah; Antoniou, Antonis C.; Jenkins, Mark ...
Human mutation,
06/2005, Volume:
25, Issue:
6
Journal Article
We describe new high-resolution observations at 843 MHz, 5 and 8.4 GHz of the galactic supernova remnant G291.0–0.1. The emission from the remnant is dominated by an elongated centrally bright ...component surrounded by weaker extended emission and subsidiary peaks. Polarization of the main component implies an orderly magnetic field aligned along its major axis. The radio emission has a relatively flat spectrum ($S\propto\nu^{-0.29}$), in common with other filled-centre remnants. The peak of the X-ray emission lies on the major axis of the elongated radio component.
Y chromosome-specific short tandem repeat (Y-STR) analysis has become another widely accepted tool for human identification. The PowerPlex® Y System is a fluorescent multiplex that includes the 12 ...loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. This panel of markers incorporates the 9-locus European minimal haplotype (EMH) loci recommended by the International Y-STR User Group and the 11-locus set recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Described here are inter-laboratory results from 17 developmental validation studies of the PowerPlex® Y System and include the following results: (a) samples distributed between laboratories and commercial standards produced expected and reproducible haplotypes; (b) use of common amplification and detection instruments were successfully demonstrated; (c) full profiles were obtained with standard 30 and 32 cycle amplification protocols and cycle number (24-28 cycles) could be modified to match different substrates (such as direct amplification of FTA® paper); (d) complete profiles were observed with reaction volumes from 6.25 to 50μL; (e) minimal impact was observed with variation of enzyme concentration; (f) full haplotypes were observed with 0.5× to 2× primer concentrations; however, relative yield between loci varied with concentration; (g) reduction of magnesium to 1mM (1.5mM standard) resulted in minimal amplification, while only partial loss of yield was observed with 1.25mM magnesium; (h) decreasing the annealing temperature by 2-4°C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2°C and significant locus dropout with a 4°C increase in annealing temperature; (i) amplification of individual loci with primers used in the multiplex produced the same alleles as observed with the multiplex amplification; (j) all laboratories observed full amplification with ≥125pg of male template with partial and/or complete profiles observed using 30-62.5pg of DNA; (k) analysis of ≤500ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with ≤1200-fold excess female DNA with the majority of alleles still observed with 10,000-fold excess female; (m) male/male mixtures produced full profiles from the minor contributor with 10-20-fold excess of the major contributor; (n) average stutter for each locus; (o) precision of sizing were determined; (p) human-specificity studies displayed amplification products only with some primate samples; and (q) reanalysis of 102 non-probative casework samples from 65 cases produced results consistent with original findings and in some instances additional identification of a minor male contributor to a male/female mixture was obtained. In general, the PowerPlex® Y System was shown to have the sensitivity, specificity and reliability required for forensic DNA analysis.
Supernova remnants with jets KESTEVEN, M. J; CASWELL, J. L; ROGER, R. S ...
Australian journal of physics,
1987, 1987-00-00, 19870101, Volume:
40, Issue:
6
Journal Article
Peer reviewed
Open access
Two examples are given of probable radio jet/supernova remnant associations: G332.4+0 1 and G 315 8 - O O. In both cases the jet length is larger than the radius of the remnant's shell, and the jet ...diameter is barely resolved and substantially less than the observed shell thickness. The jet luminosity is 5-10% that of the shell. The G332 4 +0.1 jet terminates 'in an extended plume whose luminosity is about 50% that of the shell.
A report is presented detailing observations of supernova remnants (SNRs) with the Fleurs and Molonglo synthesis radio telescopes. Fifty-four remnants have been mapped with at least one of these ...instruments, eleven with both. Approximately half of the maps have been published and a key to these publications is given.
The synthesis telescopes at Fleurs and Molonglo have been used to map 50 supernova remnants. Additional specialized software to process the maps has been developed, and Parkes observations have been ...used to supply short spacing information missing from the maps.
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